Promoters and gene expression method by using the promoters

ABSTRACT

To provide a promoter, a recombinant DNA, a gene expression vector, an expression vector, and a transformant, which are capable of expressing a gene without inducing gene expression with an inducer; and a method for producing a protein and a kit therefor, which can be operated easily and performed inexpensively by convenient and inexpensive steps. An isolated DNA having the nucleotide sequence of SEQ ID NO: 1 or 2 of Sequence Listing or a fragment thereof, wherein the isolated DNA exhibits a constitutive promoter activity in  Escherichia coli  or a bacterium belonging to the genus  Bacillus ; an isolated DNA having a nucleotide sequence of a nucleic acid capable of hybridizing to the above DNA, wherein the isolated DNA exhibits a constitutive promoter activity in  Escherichia coli  or a bacterium belonging to the genus  Bacillus ; a recombinant DNA comprising the above DNA and a foreign gene, wherein the foreign gene is operably located; a gene expression vector, at least comprising the above DNA; an expression vector comprising the recombinant DNA; a transformant having the above recombinant DNA, or the above expression vector; a method for producing a protein, characterized by culturing the above transformant, and collecting a protein from the resulting culture; and a kit for producing a protein, at least comprising the above DNA, or the above gene expression vector.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP01/03607 which has an Internationalfiling date of Apr. 26, 2001, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a novel promoter capable ofconveniently and inexpensively expressing a desired gene product at ahigh level, and concretely to an isolated DNA having a sequence of thepromoter and a method for producing a protein using the DNA.

BACKGROUND ART

When a useful gene product is produced in a genetically engineeringmanner, depending upon its purpose, there has been used an expressionsystem using as a host a microbial cell such as Escherichia coli,Bacillus subtilus, or an yeast; an animal cell, an insect cell, a plantcell or the like of which cultivation means has been established, andusing a promoter appropriate for the host. Among them, an expressionsystem using Escherichia coli as a host and lac promoter or a derivativethereof or the like is a kind of system which is often used from theviewpoint of its facility in handling.

However, the expression system using lac promoter or a derivativethereof has some defects such that the expression system is industriallydisadvantageous because the induction of gene expression is necessitatedduring expression of the gene product. For instance, lac promoter, tacpromoter or the like has some defects such that it is disadvantageous tocarry out the above expression on an industrial scale, because expensiveIPTG (isopropyl-β-D-thiogalactopyranoside) is required for the inductionof gene expression.

On the other hand, an expression vector utilizing temperature inductionof phage λ promoter has also been generally used. However, intemperature-induced overexpression of a recombinant gene product, theremay be some disadvantages of (a) difficulty for achieving a rapidshift-up of temperature; (b) increase in possibility of the formation ofinsoluble inclusion bodies at a higher culturing temperature; and (c)induction of some proteases in Escherichia coli during heat shock.

Therefore, there has been desired a means which allows highly efficientexpression without inducing gene expression.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a DNA, a recombinantDNA, a gene expression vector, an expression vector, and a transformant,all of which have promoter sequence capable of expressing a gene at ahigh level without inducing gene expression by an inducer; and a methodfor producing a protein and a kit therefor, which can be operated easilyand performed inexpensively by convenient and inexpensive steps.

The gist of the present invention relates to:

-   [1] an isolated DNA having a nucleotide sequence selected from the    group consisting of:    -   (A) the nucleotide sequence of SEQ ID NO: 1 or 2;    -   (B) a nucleotide sequence having substitution, deletion or        addition of at least one residue in SEQ ID NO: 1 or 2;    -   (C) a nucleotide sequence of a nucleic acid capable of        hybridizing to a nucleic acid consisting of the nucleotide        sequence of SEQ ID NO: 1 or 2 under stringent conditions; and    -   (D) a nucleotide sequence having at least 80% sequence identity        to the nucleotide sequence of SEQ ID NO: 1 or 2, or a fragment        thereof, wherein the isolated DNA exhibits a constitutive        promoter activity in Escherichia coli or a bacterium belonging        to the genus Bacillus;-   [2] the isolated DNA according to item [1] above, wherein the    isolated DNA is capable of expressing a foreign gene in the absence    of an inducer, when located upstream from the gene;-   [3] a recombinant DNA comprising the DNA of item [1] or [2] above    and a foreign gene, wherein the foreign gene is operably located;-   [4] the recombinant DNA according to item [3] above, wherein the    foreign gene is a nucleic acid selected from the group consisting of    a nucleic acid encoding a protein, a nucleic acid encoding antisense    RNA, and a nucleic acid encoding a ribozyme;-   [5] a gene expression vector, at least comprising the DNA of item    [1] or [2] above;-   [6] the gene expression vector according to item [5] above, wherein    the vector is a vector selected from the group consisting of plasmid    vectors, phage vectors and viral vectors;-   [7] the gene expression vector according to item [5] or [6] above,    which comprises the nucleotide sequence of SEQ ID NO: 3 or 4.-   [8] an expression vector comprising the recombinant DNA of item [3]    or [4] above;-   [9] the expression vector according to item [8] above, wherein the    vector is a vector selected from the group consisting of plasmid    vectors, phage vectors and viral vectors;-   [10] a transformant having the recombinant DNA of item [3] or [4]    above, or the expression vector of item [8] or [9] above;-   [11] a method for producing a protein, characterized by culturing    the transformant of item [10] above, and collecting a protein from    the resulting culture; and-   [12] a kit for producing a protein, at least comprising the DNA of    item [1] or [2] above, or the gene expression vector of any one of    items [5] to [7] above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing SDS-PAGE results of a crude enzymesolution and a purified enzyme of a strain screened for D-AAT activity.

FIG. 2 is a view showing the results of restriction analysis of D-AATgene from Bacillus sp. SK-1.

FIG. 3 is a view showing the results of SDS-PAGE analysis of D-AAT geneproduct from Bacillus sp. SK-1.

FIG. 4 is a schematic view of a construction of the gene expressionvector pHCE19T(II). HCE II (N) SEQ ID NO:5. HCE II (C:T7)=SEQ ID NO:6.

FIG. 5 is a schematic view showing the gene expression vectorpHCE19T(II). MCS Sequence=SEQ ID NO:10.

FIG. 6 is a schematic view of a construction of the gene expressionvector pHCE19(II). HCE II (N)=SEQ ID NO:5. HCE II (C:original)=SEQ IDNO:7.

FIG. 7 is a schematic view showing the gene expression vectorpHCE19(II). MCS Sequence=SEQ ID NO:10.

FIG. 8 is a schematic view of a construction of the expression vectorpHCE19T(II)-TNA for expressing TNA.

FIG. 9 is a view showing the results of SDS-PAGE analysis of expressionof TNA in Escherichia coli harboring the expression vectorpHCE19T(II)-TNA.

FIG. 10 is a graph showing the analytical results of growth curve ofEscherichia coli HB101/pHCE19T(II)-TNA, the synthesizing activity forTNA and the expression product, and showing a typical profile for batchculture of Escherichia coli HB101/pHCE19T(II)-TNA at 37° C. in a 2.5 Lfermenter.

FIG. 11 is a graph each showing effects of temperature and pH on thesynthesizing activity for TNA.

FIG. 12 is a graph each showing effects of ammonium chlorideconcentration, indol concentration and sodium pyruvate concentration onthe synthesizing activity for TNA.

FIG. 13 is a graph showing effects of Triton X-100 on the synthesizingactivity for TNA.

FIG. 14 is a view showing a reaction pattern of L-tryptophan productionin an enzyme reactor.

BEST MODE FOR CARRYING OUT THE INVENTION

The DNA of the present invention is localized upstream from ORF ofD-amino acid aminotransferase from Bacillus sp. SK-1, and is derivedfrom an element showing a promoter activity. The present invention isbased on a surprising finding by the present inventors that when aforeign gene (also referred to as “desired gene”) is located downstreamfrom the above-mentioned DNA, the desired gene product can be expressedat a level of at least about 30 to about 50% of total amounts ofproteins in the absence of an expression inducer.

The DNA of the present invention includes a DNA having the nucleotidesequence of SEQ ID NO: 1 or 2. More preferred are isolated DNAs eachexhibiting a constitutive promoter activity in Escherichia coli or abacterium belonging to the genus Bacillus.

In the present specification, the term “promoter” includes to the TATAbox or a TATA box-like region, which is located 20 to 30 base pairsupstream from the transcription initiation point (+1) and functions toallow RNA polymerase to initiate transcription from an accurateposition, but is not necessarily limited to portions near these regionsand may include, in addition to the regions, a region required forassociation of a protein other than the RNA polymerase for regulation ofexpression. Also, there may be described as “promoter region” in thepresent specification, and this term refers to a region containing thepromoter in the present specification.

In the present specification, the term “promoter activity” shows that agene has ability and function for producing an expression product of thegene inside or outside of the host, when the gene is located downstreamfrom the promoter in a manner such that the gene is expressible, and theresulting construct is introduced into a host.

Generally, the above-mentioned “promoter activity” can be assayed by aprocess comprising the following steps of:

-   1) ligating the DNA to be determined, to upstream region of a gene    encoding a protein which is readily quantified or confirmed    (hereinafter also referred to as a “reporter gene”);-   2) introducing the resulting construct into a host;-   3) culturing the resulting transformant, thereby expressing the    protein; and-   4) determining an expression level of the protein.    In addition, the presence or absence of the “promoter activity” can    be determined by, for instance, confirmation of expression of the    gene product of the gene inside or outside of the host, when a    sequence thought to have a promoter sequence is ligated upstream    region from he reporter gene, and the resulting construct is    introduced into a host. Here, the case where expression is found is    an index such that the promoter has a promoter activity in the    introduced host.

In the present specification, the term “constitutive promoter” refers toa promoter by which transcription is performed constantly at a givenlevel irrelevant to the growth conditions. In other words, the“constitutive promoter” is capable of expressing a gene locateddownstream from the promoter without induction by using an inducer asrepresented by IPTG or the like.

The DNA of the present invention encompasses a fragment of theabove-mentioned isolated DNA having the nucleotide sequence of SEQ IDNO: 1 or 2, as long as the constitutive promoter activity is found.Here, the “fragment” can be appropriately selected within the range sothat the constitutive promoter activity is found. The fragment can beselected from the above-mentioned process of the steps 1) to 4). Thelength of the above-mentioned “fragment” is, for instance, exemplifiedby those having 500 bases or less.

The DNA of the present invention also encompasses a DNA having anucleotide sequence having substitution, deletion or addition of atleast one base, concretely, one or more bases in the nucleotide sequenceof SEQ ID NO: 1 or 2, wherein the DNA has the constitutive promoteractivity. Generally, in DNA having a short sequence, although a DNAhaving mutation (substitution, deletion or addition) in at least onebase may show change in its activity, the present invention encompasses“a DNA having mutation” of which constitutive promoter activity is foundby the above-mentioned process of the steps 1) to 4). This mutation maybe any of naturally occurring mutations and artificially introducedmutations.

As a method for artificially introducing a mutation, there is includedconventional site-directed mutagenesis method. As the site-directedmutagenesis method, there can be employed, for instance, a methodutilizing amber mutation [gapped duplex method, Nucleic Acids Research,12, pp9441–9456 (1984)]; a method utilizing a host deficient dut(dUTPase) gene and ung (uracil-DNA glycosilase) gene [Kunkel method,Proceedings of the National Academy of Sciences of the USA, 82,pp488–492 (1985)]; a method by PCR utilizing amber mutation (WO98/02535); and the like.

The length of the DNA having mutation may be any length in which theconstitutive promoter activity is found by the above-mentioned processof the steps 1) to 4), and is exemplified by, for instance, those having500 bases or less.

In addition, the present invention encompasses the DNA (a) capable ofhybridizing with a strand complementary to the DNA of the presentinvention under stringent conditions, wherein the DNA has theconstitutive promoter activity; or the DNA (b) obtained by using, forinstance, oligonucleotide probes or primers which are designed by aconventional method on the basis of the DNA of the present invention andchemically synthesized, preferably an isolated DNA having theconstitutive promoter activity in Escherichia coli or a bacteriumbelonging to the genus Bacillus. As the DNAs, for instance, there may beselected those in which the constitutive promoter activity is found bythe above-mentioned process of the steps 1) to 4). The nucleotidesequence of the oligonucleotide probe is not particularly limited, aslong as they are those which are capable of hybridizing to theabove-mentioned DNA, or to a DNA having a nucleotide sequencecomplementary thereto under stringent conditions.

Here, the term “stringent conditions” refers to, for instance, thefollowing conditions. Concretely, in the case of the above-mentioned DNA(a), there are exemplified conditions of carrying out incubation at 50°C. for overnight in a solution containing 6×SSC (wherein 1×SSC is 0.15 MNaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5× Denhardt's [0.1%bovine serum albumin (BSA), 0.1% polyvinyl pyrrolidone, 0.1% Ficol 400],and 100 μg/ml salmon sperm DNA. In the case of the above-mentioned DNA(b), there are exemplified conditions of carrying out incubationovernight at a temperature of Tm of the probe used minus(−) 25° C. inthe solution similarly prepared as mentioned above.

Also, the nucleotide sequence of the primer is not particularly limited,as long as the primer is annealed to the above-mentioned DNA or a DNAhaving a nucleotide sequence complementary thereto and allows the DNApolymerase to initiate the extension reaction under usual reactionconditions for PCR.

Tm of the oligonucleotide probe or primer can be calculated, forinstance, by the following equation:Tm=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)wherein N is a chain length of the oligonucleotide probe or primer; and% G+C is a content of guanine and cytosine residues in theoligonucleotide probe or primer.

In addition, when the chain length of the oligonucleotide probe orprimer is shorter than 18 bases, Tm can be deduced from a sum of aproduct of the contents of A+T (adenine+thymine) residues multiplied by2° C., and a product of the contents of G+C residues multiplied by 4°C., [(A+T)×2+(G+C)×4].

The chain length of the above-mentioned oligonucleotide probe is notparticularly limited. It is preferable that the chain length is 15 basesor more, more preferably 18 bases or more, from the viewpoint ofpreventing nonspecific hybridization.

Also, the chain length of the primer is not particularly limited. Forinstance, it is preferable that the chain length is 15 to 40 bases,preferably 17 to 30 bases. The above-mentioned primer can be used forvarious gene amplification methods such as PCR method, and the resultingDNA having the constitutive promoter activity is also encompassed in thepresent invention.

Also, the details of the hybridization manipulations are described, forinstance, in Molecular Cloning: A Laboratory Manual, 2nd Ed., publishedby Cold Spring Harbor Laboratory in 1989, edited by T. Maniatis et al.

The length of the above-mentioned DNAs (a) and (b) may be a length inwhich the constitutive promoter activity is found by the above-mentionedprocess of the steps 1) to 4). For instance, there may be exemplifiedthose having a length of 500 bases or less.

Also, the DNA of the present invention encompasses a DNA having anucleotide sequence having at least 80% sequence identity, preferably atleast 90% sequence identity, more preferably at least 95% sequenceidentity to the nucleotide sequence of SEQ ID NO: 1 or 2, wherein theDNA has the constitutive promoter activity. More concretely, a DNAhaving at least 80% sequence identity, preferably at least 90% sequenceidentity, more preferably at least 95% sequence identity to thenucleotide sequence of SEQ ID NO: 1, wherein the DNA has theconstitutive promoter activity; a DNA having at least 80% sequenceidentity, preferably at least 90% sequence identity, more preferably atleast 95% sequence identity to the nucleotide sequence of SEQ ID NO: 2,wherein the DNA has the constitutive promoter activity; and the like.This DNA may be, for instance, any of those in which the constitutivepromoter activity is found by the above-mentioned process of thesteps 1) to 4).

The above-mentioned “sequence identity” refers to sequence similarityfor residues between the two polynucleotides. The above-mentioned“sequence identity” can be determined by comparing two nucleotidesequences aligned optimally over the region of the nucleotide sequenceto be compared. Here, the polynucleotide to be compared may haveaddition or deletion (for instance, gap, overhang, or the like) ascompared to the reference sequence (for instance, consensus sequence orthe like) for optimally aligning the two sequences.

The numerical value (percentage) of the sequence identity can becalculated by determining identical nucleic acid bases which are presentin both of the sequences to determine the number of matched sites,thereafter dividing the above-mentioned number of the matched site by atotal number of bases present within the region of the sequence to becompared, and multiplying the resulting numerical value by 100. Analgorithm for obtaining optimal alignment and homology includes, forinstance, local homology algorithm by Smith et al. [Add. APL. Math., 2,p482 (1981)], homology alignment algorithm by Needleman et al. [Journalof Molecular Biology, 48, p443, (1970)], and homology searching methodby Pearson et al. [Proceedings of the National Academy of Sciences ofthe USA, 85, p2444 (1988)]. More concretely, there are included dynamicprogramming method, gap penalty method, Smith-Waterman algorithm,Good-Kanehisa algorithm, BLAST algorithm, FASTA algorithm, and the like.

The sequence identity between the nucleic acids is determined, forinstance, by using a sequence analysis software, concretely BLASTN,FASTA and the like. The above-mentioned BLASTN is generally accessibleat homepage address: http://www.ncbi.nlm.nih.gov/BLAST/, and theabove-mentioned FASTA is generally accessible at homepage address:http://www.ddbj.nig.ac.jp.

According to the DNA of the present invention, there is also provided arecombinant DNA comprising the DNA of the present invention and aforeign gene, wherein the foreign gene is operably located. Therecombinant DNA is also encompassed in the present invention.

In the present specification, a foreign gene is intended to encompassany of a gene derived from a different origin from the organism fromwhich the DNA of the present invention is originated; a geneheterogenous to the DNA of the present invention; or a gene heterogenousto a host when the DNA of the present invention is used by introducingan appropriate host as described below.

The above-mentioned foreign gene is not particularly limited. Theforeign gene includes, for instance, a nucleic acid encoding a protein,a nucleic acid encoding antisense RNA, a nucleic acid encoding aribozyme, and the like. The origin of the foreign gene is notparticularly limited. The origin includes, for instance, microorganismssuch as bacteria, yeasts, Actinomycetes, filamentous fungi, Ascomycetes,and Basidiomycetes; plants; insects; animals; and the like, and thereare also included artificially synthesized genes according to itspurpose.

The nucleic acid encoding a protein includes, for instance, nucleicacids encoding enzymes, cytokines, antibodies, and the like. Moreconcretely, the nucleic acid includes, for instance, interleukin 1–12genes, interferon α, β or γ gene, tumor necrosis factor gene,colony-stimulating factor gene, erythropoietin gene, transforming growthfactor-β gene, immunoglobulin gene, tissue plasminogen activator gene,urokinase gene, firefly luciferase gene, and the like, without beinglimited thereto.

The “ribozyme” in the present specification refers to a substance whichcleaves mRNA for particular proteins, and inhibits translation for theseparticular proteins. The ribozyme can be designed from the sequence of agene encoding the particular protein. For instance, hammerhead-typeribozyme can be prepared by the method described in FEBS Letter, 228,pp228–230 (1988). Also, in addition to the hammerhead-type ribozyme, theribozyme in the present specification encompasses any of those capableof cleaving mRNA for particular proteins and inhibiting translation ofthese particular proteins, regardless of the kinds of ribozymes such ashairpin-type ribozymes and delta-type ribozymes.

In the present specification, “the foreign gene is operably (located)”means that the foreign gene is under control of the promoter activityexhibited by the DNA of the present invention.

The method for obtaining a DNA of the present invention includes, forinstance, (1) a method of isolation from an organism, as described inExamples set forth below; (2) a method of selection from genomic DNA ofan organism using appropriate probes or primers, which are based on thenucleotide sequence of SEQ ID NO: 1 or 2; (3) a chemically synthesizingmethod on the basis of the nucleotide sequence of SEQ ID NO: 1 or 2; andthe like.

When a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 2 isprepared by chemical synthesis, the above-mentioned DNA can besynthesized by, for instance, enzymatically ligating oligonucleotidessynthesized chemically by conventional phosphoramidite method or thelike. Concretely, the DNA can be obtained by, for instance, thefollowing steps:

-   (1) synthesizing each of several dozen kinds of oligonucleotides    A_(n) (wherein n is a positive integer) which can cover the    nucleotide sequence of SEQ ID NO: 1 or 2, and-    complementary strand oligonucleotides a_(n) (wherein n is a    positive integer) having the same length as A_(n), and consisting of    a sequence complementary to a nucleotide sequence resulting from a    shift with several bases at 3′-side or 5′-side on the A_(n)    sequence, wherein the complementary strand oligonucleotides an are    annealed with the oligonucleotides A_(n), thereby generating    double-stranded DNAs having 5′-cohesive end or 3′-cohesive end, by    conventional chemical synthesis methods    -   [For instance, when an oligonucleotide consisting of the        nucleotide sequence of SEQ ID NO: 1 is prepared, the        above-mentioned A_(n) are oligonucleotides each consisting of        base nos: 1–20 (A₁), 21–40 (A₂), 41–60 (A₃), 61–80 (A4), 81–100        (A₅), 101–120 (A6), 121–140 (A₇), 141–160 (A₈), 161–180 (A₉),        181–200 (A₁₀), and 201–223 (A₁₁) of SEQ ID NO: 1, and    -    the above-mentioned a_(n) are strands complementary to        oligonucleotides each consisting of base nos: 1–23 (a₁), 24–43        (a₂), 44–63 (a₃), 64–83 (a₄), 84–103 (a₅), 104–123 (a₆), 124–143        (a₇), 144–163 (a8), 164–183 (a9), 184-203 (a₁₀), and 204-223        (a₁₁) of SEQ ID NO: 1.];-   (2) phosphorylating each 5′-end of the oligonucleotides A_(n) and    each 5′-end of the corresponding complementary strand    oligonucleotides a_(n), obtained in the step (1) mentioned above, by    using ATP and conventional T4 polynucleotide kinase;-   (3) annealing each of the oligonucleotides A_(n) and each of the    corresponding complementary strand oligonucleotides a_(n), obtained    in the step (2) mentioned above, thereby giving several dozens of    “double stranded DNA (A_(n)a_(n)) having 5′-cohesive end or    3′-cohesive end”;-   (4) dividing the double-stranded DNAs (A_(n)a_(n)) obtained in the    step (3) mentioned above into several blocks corresponding to the    nucleotide sequence of SEQ ID NO: 1 or 2 in an n-ascending order,    and putting the double-stranded DNAs (A_(n)a_(n)) obtained in the    step (3) mentioned above together in one tube corresponding to each    block    -   [For instance, when the oligonucleotide consisting of the        nucleotide sequence of SEQ ID NO: 1 is prepared, there are set:    -   a tube for oligonucleotides A₁–A₄ and complementary strands        a₁–a₄,    -   a tube for oligonucleotides A₅–A₈ and complementary strands        a₅–a₈, and    -   a tube for oligonucleotides A₉–A₁₁ and complementary strands        a₉–a₁₁.];-   (5) ligating the double-stranded DNAs for every tube in the step (4)    mentioned above corresponding to each block, thereby giving    double-stranded DNAs corresponding to each block;-   (6) subjecting the double-stranded DNAs obtained in the step (5)    mentioned above to gel electrophoresis, and extracting from a gel a    band for the double-stranded DNA having the desired chain length    corresponding to each block;-   (7) putting together and ligating the double-stranded DNAs each    having the desired chain length corresponding to each block obtained    in the step (6) mentioned above; and-   (8) examining the chain length on gel electrophoresis and/or    performing nucleotide sequencing for the DNA obtained in the    step (7) mentioned above, thereby confirming that the resulting DNA    is DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 2.

Since the DNA of the present invention is a promoter which is capable ofexpressing the gene at a high level without inducing gene expression,the DNA is especially suitable for expression of a foreign gene, whichis a nucleic acid encoding a protein.

Also, according to the DNA of the present invention, there is provided agene expression vector at least comprising the DNA. The gene expressionvector is also encompassed in the present invention.

According to the gene expression vector, since the vector comprises theDNA of the present invention, the desired gene product can beconstitutively expressed on the level of at least about 30 to about 50%of total amounts of intracellular protein produced by the host, and thedesired gene product can be easily expressed depending upon its purposesand the like.

Concrete examples of the gene expression vector of the present inventioninclude pHCE19T(II) [FIG. 5], and pHCE19(II) [FIG. 7].

Incidentally, Escherichia coli JM109 transformed with theabove-mentioned plasmid pHCE19T(II) was named and represented asEscherichia coli JM109/pHCE19T(II) and deposited under the BudapestTreaty with the accession number of FERM BP-7535 with the InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, of which the address is AIST Tsukuba Central 6,1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-8566, Japan, sinceApr. 14, 2000 (original date of deposit). Also, Escherichia coli JM109transformed with the above-mentioned plasmid pHCE19 (II) was named andrepresented as Escherichia coli JM109/pHCE19(II) and deposited under theBudapest Treaty with the accession number of FERM BP-7534 with theInternational Patent Organism Depositary, National Institute of AdvancedScience and Technology, of which the address is AIST Tsukuba Central 6,1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-8566, Japan, sinceApr. 14, 2000 (original date of deposit). pHCE19T(II) and pHCE19(II),which are one of examples of the gene expression vectors of the presentinvention can also be made available from deposited Escherichia coli.

In the gene expression vector of the present invention, a vectorincludes plasmid vectors, phage vectors, and viral vectors. Theabove-mentioned vector can be appropriately selected depending upon thecell which is used as a host.

The cell which can be used as a host includes Escherichia coli or abacterium belonging to the genus Bacillus. Concretely, the Escherichiacoli includes HB101 strain, C600 strain, JM109 strain, DH5α strain, DH10B strain, XL-1BlueMRF' strain, TOP10F strain and the like of the lineageof Escherichia coli K-12. Also, the bacterium belonging to the genusBacillus includes Bacillus sp., Bacillus subtilis, Bacillusstearothermophilus, and the like, without being limited thereto. Thoseobtained by mutation treatment and the like of these Escherichia coli orbacteria belonging to the genus Bacillus can also be used as a host.

The above-mentioned vector includes, for instance, when the host isEscherichia coli, a plasmid vector including pUC18, pUC19, pBluescript,pET, and the like, and a phage vector including lambda phage vectorssuch as λgt10 and λgt11, and the like.

For the construction of the gene expression vector of the presentinvention, there can be utilized techniques described in theabove-mentioned Molecular Cloning: A Laboratory Manual, 2nd Ed., and thelike. For instance, the vector can be prepared in accordance with thescheme for the construction shown in FIG. 4. In other words, a DNAfragment (350 bp) comprising 5′-promoter located upstream region ofD-AAT gene from thermophilic Bacillus sp. SK-1 and SD (Shine-dalgano)sequence is amplified by PCR method using the following primers [primer1 (SEQ ID NO: 5) and primer 2 (SEQ ID NO: 6)]:

-   primer 1 (5′ccaagcttgatctctccttcacagattcc-3′)(29 mer) and-   primer 2 (5′gaggatccagccatggtatatctcctttttccagaagtgtgaaa-3 ′)(44    mer)

Next, the amplified fragment containing promoter is digested withHindIII and BamHI, and the resulting product is purified. The resultingfragment is ligated to a larger fragment of HindIII- and BamHI-digestedpUC19, to prepare pHUC19T. Subsequently, an NcoI-ScaI fragmentcontaining potent transcription terminator rrnBT1T2 derived from anexpression vector pTrc99A is inserted into an NcoI-EcoRI (filling-in)site on pHUC19T, whereby a gene expression vector pHCE19T(II) can beobtained (FIG. 5). The sequence of the promoter region can be confirmedby DNA nucleotide sequencing. Also, the same procedures are carried outusing the following primer 3 (SEQ ID NO: 5):

-   primer 3 (5′-agggatccagccatgggttccagctcctttttccagaa-3′)(38 mer)-   in place of primer 2, whereby the gene expression vector pHCE19(II)    having a length different from that between SD sequence and NcoI    site can be obtained (FIGS. 6 and 7).

Also, the above-mentioned pHCE19(II) can be obtained by mutating thenucleotide sequence: 5′-gatata-3′, which is located between SD sequenceand NcoI site of pHCE19T(II), to 5′-gctggaac-3′ by known mutagenesismethod.

The gene expression vector of the present invention may contain aterminator such as rrnBT1T, a selectable marker gene, and the like.

The selectable marker includes ampicillin-resistant gene,kanamycine-resistant gene, chloramphenicol-resistant gene, and the like.

In addition, the gene expression vector of the present invention maycontain, depending on its purpose of the desired gene product, forinstance, for simplification of the isolation procedure of the desiredgene product, a sequence which allows to be expressed as a fusionprotein with a heterologous protein such as glutathione-S-transferaseand maltose-binding protein, or a tag sequence which allows to beexpressed as a protein to which histidine tag or the like is added.

The gene expression vector of the present invention includes, forinstance, a vector having the nucleotide sequence of SEQ ID NO: 3 or 4.

Further, according to the present invention, there is provided anexpression vector comprising the above-mentioned recombinant DNA. Theexpression vector is also encompassed in the present invention. Inaddition, the expression vector of the present invention encompasses aconstruct obtained by incorporating a desired gene into theabove-mentioned gene expression vector.

The same vectors as those vectors listed as the above-mentioned geneexpression vector can be used.

The expression vector of the present invention can be prepared by (a)incorporating the above-mentioned recombinant DNA into an appropriatevector, and (b) incorporating a desired gene to the above-mentioned geneexpression vector.

Also, according to the recombinant DNA and the expression vector of thepresent invention, there can be provided a transformant harboring theabove-mentioned recombinant DNA, or a transformant harboring theabove-mentioned expression vector.

The host includes the same cells as the previously mentioned “cell(s)which can be used as a host.”

The introduction of the recombinant DNA into a host can be carried out,for instance, by the methods described in the literatures [Virology, 52,p456 (1873), Molecular and Cellular Biology, 7, p2745 (1987), Journal ofthe National Cancer Institute, 41, p351 (1968), EMBO Journal, 1, p841(1982)].

The introduction of the expression vector into a host can be carriedout, for instance, by calcium phosphate method [Molecular and CellularBiology, 7, p2745 (1987)], electroporation method [Proceedings of theNational Academy of Sciences of the USA, 81, p7161 (1984)], DEAE-dextranmethod [Methods in Nucleic Acids Research, p283, Edit. by Callam,published by CRC Press, 1991], liposome method [BioTechniques, 6, p682(1989)]; and the like.

Further, according to the transformant of the present invention, thereis provided a method for producing a protein, characterized by culturingthe transformant, and collecting a protein from the resulting culture.This “method for producing a protein” is also encompassed in the presentinvention.

More concretely, a protein can be produced by the steps of:

-   (I) transforming a host cell using:    -   a) a recombinant DNA in which a nucleic acid encoding a protein        is located downstream from the DNA of the present invention in        an expressible state; or    -   b) a vector comprising the recombinant DNA; and-   (II) culturing the transformant obtained in (I) under conditions    appropriate for expression of a protein, and collecting the protein    from the culture.

The culture conditions of the transformant can be appropriately selecteddepending upon the characteristics of the cell used as a host and theprotein to be expressed.

The resulting protein can be purified by a conventional proteinpurification means. The purification means include, for instance,salting-out, ion exchange chromatography, hydrophobic chromatography,affinity chromatography, gel filtration chromatography, and the like.

In the method for producing a protein of the present invention, therecan be used a kit for producing a protein, comprising the DNA of thepresent invention or the gene expression vector of the presentinvention. According to the kit, the method for producing a protein canbe more conveniently carried out.

The present invention will be described in detail by means of Examples,without intending to limit the present invention thereto.

EXAMPLE 1 Screening and Characterization of D-Amino AcidAminotransferase-Producing Thermophilic Bacteria

In order to obtain biocatalysts useful for enzymatic synthesis ofD-amino acid and industrial production of D-amino acid, D-amino acidaminotransferase activity (D-AAT) was analyzed for the 1,300 kinds ofthermophilic bacteria isolated from the soil of Korea.

The D-ATT activity was determined by measuring the rate of pyruvateformation from D-alanine and α-ketoglutarate. The amount of pyruvate wasdetermined by the coupling method with lactate dehydrogenase or by thesalycylaldehyde method [Analytical Biochemistry, 27, pp1659–1660(1995);Methods in Enzymology, 113, p108(1985)].

In a determination of the activity, a reaction mixture containing 50 mMpyridoxal-5′-phosphate, 100 mM D-alanine and 100 mM α-ketoglutarate in100 mM Tris-HCI (pH 8.5) was used. In the case of lactate dehydrogenase(LDH)-coupling assay, 0.3 mM NADH and 5 units/ml LDH were includedthereto. One unit of the enzyme activity was defined as the amount ofthe enzyme catalyzing the formation of 1 mmol of pyruvate per minute.The protein concentration was determined by means of the Bradford method[Analytical Biochemistry, 72, pp248–254 (1976)] using bovine serumalbumin as a standard.

As a result of searching for the D-AAT activity derived fromthermophilic bacteria isolated from various environments, 110 strains ofD-AAT-producing bacteria were obtained. Most of them were thought tobelong to the genus Bacillus, since they were grown under aerobicconditions and formed endospores.

Subsequently, the D-AAT-producing strain was compared for their enzymeactivity and thermal stability. As a result, four thermophilic bacteria:two thermophilic bacteria each having higher specific activity, Bacillussp. LK-1 and LK-2, and two thermophilic bacteria each having higherthermal stability than the above-mentioned D-ATT-producing strains,Bacillus stearothermophilus KL-01 and Bacillus sp. SK-1 were selected asthe strains producing proteinous biocatalysts for production of D-aminoacid. Two strains LK1 and LK2 each showing the highest D-AAT activity inthe D-AAT activity-positive strains, were subjected to taxonomicidentification, and it is found that the morphological and biochemicalcharacteristics of the above-mentioned strains were shown to be verysimilar to those of YM1 strain [Kor. J. Microbiol. Biotechnol., 27,pp184–190 (1999)]. In particular, unlike YM1 strain, the above-mentionedtwo strains could not grow at 65° C. In Western blot analysis of D-AATsderived from different Bacillus, the LK1 enzyme and LK2 enzyme showedstrong interaction with the antibody against YM1 D-AAT, and themobilities of the two isolated D-AATs are the same as that of the D-AATderived from YM1 (molecular weight: approximately 31 kDa) (FIG. 1).

On the other hand, D-AATs derived from the thermophilic Bacillus strainscapable of growing at 65° C. showed relative weak immunoblot signals,and the mobility of D-AAT was clearly distinct from that of YM1 D-AAT,which showed molecular weight of 32–34 kDa on SDS-polyacrylamide gelelectrophoresis analysis (FIG. 1). The D-AAT activity of the Bacillusstrains capable of growing at 65° C. was less than 0.02 units/mgprotein, which was approximately one-fifth of the D-AAT activities eachof LK1 and LK2 [Kor. J. Microbiol. Biotechnol., 27, pp184–190 (1999)].In order to clarify the characteristics of D-AATs derived from Bacillusgroup capable of growing at 65° C., in this experiment, SK-1 strain wasselected as the source of D-AAT.

EXAMPLE 2 Characterization of Thermostable D-Amino AcidAminotransferase-Producing Thermophilic Bacillus sp. SK-1

Isolated Bacillus sp. SK-1 had an obligately symbiotic interaction withSymbiobacterium toebii. The characteristics of the above-mentioned SK-1strain are shown as follows:

-   Bacteriological Characteristics-   Aerobic, gram-positive, a motile bacillus-   GC content of genomic DNA: 43.9 mol %-   Optimum growth conditions    -   Temperature: 45–70° C. (optimum temperature: 60° C.)    -   pH: 6.0–9.0 (optimum pH: pH 7.5)-   Presence of meso-diamino pimelic acid in the cell wall-   Presence of branched-chain fatty acids iso-15:0 and iso-17:0 as main    cellular fatty acids-   Main isoprenoid quinone: MK-7-   Comparative sequence analysis of 16S ribosomal DNA    -   Closely related to Bacillus thermoglucosidasius

EXAMPLE 3 Gene Cloning and Characterization of Thermostable D-Amino AcidAminotransferase Derived from Thermophilic Bacillus sp. SK-1

1) Bacterial Strain and Plasmids

Bacillus sp. SK-1 was cultured in MY medium [composition: 1.5%polypeptone, 0.2% yeast extract, 0.2% meat extract, 0.2% glycerol, 0.2%K₂HPO₄, 0.2% KH₂PO₄ and 0.026% NH₄Cl (pH 7.0)] under aerobic conditionsat 60° C. In order to clone the D-AAT gene, Bacillus sp. SK-1 (symbioticbacterium of obligately symbiotic thermophilic bacterium Symbiobacteriumtoebii SC-1) was used as the DNA source. Escherichia coli strain WM335[leu, pro, his, arg, thyA, met, lac, gal, rspL, hsdM, hsdR, murI] (anauxotrophic strain for D-glutamic acid) was employed as the host forgene complementation [Journal of Bacteriology, 114, pp499–506 (1978)].If necessary, Bacillus sp. SK-1 was cultured in Luria-Bertani mediumsupplemented with ampicillin (100 mg/ml) and D-glutamic acid (100mg/ml).

Plasmids pBluescript II SK and pBluescript II KS were purchased fromStratagene. Plasmids pUC118 and pUC119 were purchased from Takara ShuzoCo. Ltd. Escherichia coli XL1-Blue (manufactured by Stratagene) was usedas a host strain for subcloning and sequencing of the D-AAT gene.

2) Gene Cloning of D-AAT Derived from Bacillus sp. SK-1

Genomic DNA of Bacillus sp. SK-1 was prepared as described by Saito etal. [Biochimica et Biophysica Acta, 72, pp619–629 (1963)], andthereafter the resulting DNA was partially digested with Sau3AI. DNAfragments of 3–10 kb were isolated by centrifugation on sucrose gradient[5–40% (w/v)] in Beckman SW40 rotor at 25,000 rpm for 20 hours. Then,the resulting fragments were ligated with BamHI-digested pUC118 by usingT4 DNA ligase for 12 hours at 16° C. The resulting legation mixture wasused to transform the D-glutamate-dependent auxotrophic strain(Escherichia coli WM335) by electroporation. Thus, a genomic DNA libraryof Bacillus sp. SK-1 was obtained. From the genomic DNA library, 35colonies each showing D-glutamic acid-independency and ampicillinresistance were obtained by the complementation for WM335.

The plasmid DNA from each colony was purified by using JetStar 2.0Plasmid Midi Purification Kit (manufactured by Genomed Co.).

Using isolated plasmids, Escherichia coli XL1-Blue cells weretransformed. Thereafter, the transformants were cultured, and theresulting cells were assayed for D-AAT activity in the same manner as inExample 1.

Among of 12 clones showing D-AAT activity, one clone (pDSK2) showing thehighest D-AAT activity [35 units/mg protein (activity in 1750-foldshigher than that of cell-free extract from Bacillus sp. SK-1 showing0.02 units/mg protein)] contained a 3.6 kb insert. In order to subclonethe D-AAT gene, plasmid pDSK2 was subjected to restriction analysis. Asa result, the D-AAT activity for pDSK2 was found to be attributed to the1.7 kb EcoRI-XhoI fragment (FIG. 2). The above-mentioned 1.7 kb fragmentwas subcloned into pbluescript II SK and pbluescript II KS, to constructpDSK231 and pDSK232, respectively. The plasmid pDSK232 containing theinsert in the opposite direction to pDSK1 showed D-AAT activity at thesame level thereto, indicating that this gene was constitutivelyexpressed by its own promoter.

The nucleotide sequence of the D-AAT gene derived from Bacillus sp. SK-1was determined using the PRISM kit (manufactured by Perkin Elmer) andApplied Biosystems 373A DNA sequencer by primer walking on the both DNAstrands. Starting primers were pBluescript II SK universal primer andreverse primer, and the primers specific to the internal sequence weresynthesized by Korea Biotech (Korea). The sequences determined wereanalyzed by using Software GENETIX-MAC (S.D.S).

3) Purification

Escherichia coli XL1-Blue cells harboring pDSK2 were cultured in LBmedium (1 liter) containing 100 mg/ml ampicillin at 37° C. for 16 hours.The resulting cells were collected by centrifugation. The collectedcells subsequently were suspended in 50 ml of standard buffer [30 mMTris-HCl (pH 8.0) containing 0.01% β-mercaptoethanol in finalconcentration and 20 mM pyridoxal-5′-phosphate (PLP) in finalconcentration]. The resulting suspension was homogenized by usingSonifier 450 [manufactured by Brason Ultrasonics] on ice with 20%efficiency for 10 minutes. After centrifugation at 15,000 rpm for 60minutes, the supernatant was collected and heat-treated at 60° C. for 20minutes. Protein aggregates were removed by centrifugation at 15,000 rpmfor 10 minutes.

The resulting supernatant was subjected to Resource Q anion-exchangecolumn (manufactured by Pharmacia) equilibrated with the above-mentionedstandard buffer. Proteins were eluted with a linear gradient of 0–1 MNaCl. The fractions showing D-AAT activity were pooled and concentratedby ultrafiltration (manufactured by Amicon). The resulting proteinsolution was treated so as to contain 1.7 M ammonium sulfate and appliedto Phenyl-Superose column (manufactured by Pharmacia) equilibrated withthe standard buffer containing 1.7 M ammonium sulfate. Protein waseluted with a linear gradient of 1.7-0 M ammonium sulfate. The fractionshaving D-AAT activity were collected and concentrated. Thereafter, theresulting concentrate was dialyzed against the standard buffer. Theresulting fraction was stored in the deep freezer (at −80° C.). Allcolumn procedures were performed by the FPLC system (manufactured byPharmacia) at room temperature.

The fraction obtained from Escherichia coli XL1-Blue harboring plasmidpDSK2 revealed a very thick band of protein of a molecular weight 34 kDa(FIG. 3). The amount of the 34 kDa-band was quantified by using an imageanalyzing system (manufactured by Biorad). As a result, it was shownthat the amount of the expressed protein was approximately 60% of thetotal Escherichia coli proteins.

EXAMPLE 4 Construction of Constitutive Expression System (GeneExpression Vector)

In order to construct a gene expression vector capable of constitutivelyexpressing a foreign gene, there were used enzymes for gel purificationand plasmid purification and kits for gel purification and plasmidpurification, which were commercially available. Unless otherwisespecified, the plasmids were constructed according to conventionaltechniques as described in, for example, T. Maniatis et al. (eds.),Molecular Cloning: A Laboratory Manual, 2nd ed, and the like.

Scheme of construction of the gene expression vector pHCE19T(II) isshown in FIG. 4. A DNA fragment (350 bp) containing 5′-promoter and SD(Shine-Dalgarno) sequence of upstream region from the above-mentionedD-AAT gene from thermophilic Bacillus sp. SK-1 was amplified by PCRusing the following primers [primer 1 (SEQ ID NO: 5) and primer 2 (SEQID NO: 6)]:

-   primer 1 (5′-ccaagcttgatctctccttcacagattcc-3′)(29 mer)-   primer 2 (5′-gaggatccagccatggtatatctcctttttccagaagtgtgaaa-3′) (44    mer).

The amplified fragment containing the promoter was digested with HindIIIand BamHI, and thereafter, the resulting product was purified. Theresulting fragments were ligated to the larger fragment in HindIII- andBamHI-digested pUC19, to produce pHUC19T. Subsequently, the NcoI-ScaIfragment containing a potent transcription terminator rrnBT1T2 derivedfrom expression vector pTrc99A was inserted into the NcoI-EcoRI(filling-in) site of pHUC19T, to obtain gene expression vectorpHCE19T(II) (FIG. 5). Incidentally, the sequence of the promoter regionwas confirmed by DNA sequencing.

In addition, the gene expression vector pHCE19(II) which is 2 b longerin the length between SD sequence and the NcoI site, was obtained byusing the following primer 3 (SEQ ID NO: 7):

-   primer 3 (5′-agggatccagccatgggttccagctcctttttccagaa-3′) (38 mer)    in place of primer 2 (FIGS. 6 and 7), in the same manner as    described above.

The nucleotide sequence of the gene expression vector pHCE19T(II)produced in the described above is shown in SEQ ID NO: 3 of SequenceListing, and the 223 bp portion corresponding to the promoter regionlocated upstream from the translation initiation codon ATG is shown inSEQ ID NO: 1 of Sequence Listing.

In addition, the nucleotide sequence of gene expression vectorpHCE19(II) produced as the same manner is shown in SEQ ID NO: 4 ofSequence Listing, and the 225 bp portion corresponding to the promoterregion located upstream from the translation initiation codon ATG isshown in SEQ ID NO: 2 of Sequence Listing.

EXAMPLE 5 Large-Scale Production of Tryptophan Indole-Lyase UsingConstitutive Expression System

In this example, using the above-mentioned pHCE19T(II), large-scaleproduction in Escherichia coli cells was attempted by cloned TNA genefor tryptophan indole-lyase (hereinafter also referred to as “TNA”)derived from thermophilic bacterium Symbiobacterium toebii SC-1. Inaddition, in order to maximize the TNA production using a batch culturesystem, culture conditions for recombinant Escherichia coli cells werestudied.

1) Construction of Constitutive Expression Vector Having TNA Gene

As shown in the strategy described in FIG. 8, the constitutiveexpression vector pHCE19T(II)-TNA carrying the TNA gene derived fromSymbiobacterium toebii SC-1 was prepared using the pHCE19T(II) describedabove.

The TNA gene was amplified by PCR using as a template the pTNA carryingthe TNA gene. The primers used are as follows:

-   N-terminal primer: 5′-ccaaagggcgagccctttaa-3′(20 mer) (SEQ ID NO: 8)-   C-terminal primer: 5′-tgactaagtctgcagaagcttattagaccagatcgaagtgcgc-3    (43 mer) (SEQ ID NO: 9).

The amplified TNA gene was digested with HindIII, and thereafter, theresulting product was purified. The pHCE19T(II) was digested with NcoI,and thereafter, the ends were filled in with the Klenow fragment,followed by redigestion with HindIII. The resulting TNA gene was ligatedto the larger fragment in the above pHCE19T(II), to obtainpHCE19T(II)-TNA.

2) Preparation of Transformants

Using Gene Pulser system (manufactured by Bio-Rad), Escherichia coliHB101[supE44, hsd20(rB- mB-), recA13, ara-14, proA2, lacY1, galK2,rpsL20, xyl-5, mtI-1, leuB6, thi-1] as a host was transformed with theabove-mentioned pHCE19T(II)-TNA.

Transformants were cultured in LB medium containing 10 g/l tryptophan(manufactured by Difco), 5 g/l yeast extract (manufactured by Difco) and10 g/l sodium chloride.

3) Preparation of Enzyme Solution

The transformants obtained in the above-mentioned item 2) were collectedby centrifugation at 5,000×g for 20 minutes, and the resulting cellswere washed with 50 mM potassium phosphate buffer (pH 8.5). Theresulting cells were suspended in 50 mM potassium phosphate buffer (pH8.5) containing 0.1 mM PLP and 10 mM 4-aminophenyl-methanesulfonylfluoride. The resulting cells were disrupted by ultrasonication usingBranson Sonifier (manufactured by Branson Ultrasonics). Then, theresulting cell lysate was centrifuged at 20,000×g for 60 minutes, toremove cell debris, and the resulting supernatant was dialyzed against20 mM potassium phosphate buffer (pH 8.5) containing 0.05 mM PLP. Thesolution after dialysis was heated at 65° C. for 30 minutes, to removeheat-denatured Escherichia coli protein including tryptophanindole-lyase derived from Escherichia coli. Thereafter, the resultingproducts were cooled on ice. The protein aggregate was removed bycentrifugation at 5,000×g for 20 minutes, and the resulting supernatantwas used as an enzyme solution. The above-mentioned enzyme solution wasstored at −20° C. until use.

4) Determination of L-Tryptophan Synthesizing Activity

L-Tryptophan synthesizing activity of TNA was determined in the mixture(pH 8.5) containing 25 mM indole, 100 mM sodium pyruvate and 500 mMammonium chloride. The reaction was initiated by addition of the enzymesolution and stopped after the incubation for 20 minutes at 65° C. byaddition of 0.05 ml of 5 N HCl. L-Tryptophan generated was measured byHPLC equipped with a reverse-phase column (manufactured by Waters, tradename: μBondapak C18). Elution was performed at a flow rate of 1ml/minute using a solution containing 5% (v/v) methanol, 2%(v/v) aceticacid and 0.1 M sodium phosphate buffer (pH 6.5). Detection of proteinwas performed by an absorptiometer at 280 nm. One unit of the enzyme wasdefined as the amount of the enzyme catalyzing the synthesis of 1 μmolL-tryptophan per minute under the above-mentioned conditions. Thespecific activity was represented by units for one milligram of theenzyme. The protein concentration was determined by the Bradford methodusing bovine serum albumin (manufactured by Sigma) as the standard.

5) Production of L-Tryptophan

Production of L-tryptophan was carried out using an agitating reactor.In order to inhibit oxidization of substrate indole, the enzyme reactorwas filled with nitrogen gas. The reaction temperature was controlled at37° C. by water circulation through a water-jacket. In order to preventdepletion of the substrate, the concentration of indole was determinedby HPLC and thereafter, indole and sodium pyruvate were intermittentlysupplied to the reaction solution.

6) Growth Curve of Cells

The optical density (OD) of a cell culture was determined at 600 nmusing a absorptiometer (trade name: BioChrom 4060, manufactured byPharmacia).

7) Large-Scale Production of TNA

According to the above-mentioned items 1)-6), the production of TNA wascarried out by using Escherichia coli (HB101/pHCE19T(II)-TNA) harboringpHCE19T(II)-TNA. As a result, as shown in FIG. 9, the protein wasexpressed in the content of approximately 40% of the total cellularproteins of Escherichia coli. In order to achieve large-scale productionof TNA under the conditions in high cell growth in the batch culture,Escherichia coli HB101/pHCE19T(II)-TNA was cultured in complex mediumcontaining 50 g/l glycerol.

Profile for the batch culture of Escherichia coli HB101/pHCE19T(II)-TNAat 37° C. in a 2.5 L fermentor is shown in FIG. 10. In the figure, theopen circles indicate dry weights of the cells (g/L), open uprighttriangles indicate TNA synthesis activity (units/ml), and open invertedtriangles indicate glycerol concentrations (g/L). After three-hourculturing, TNA production was increased along with cell growth. Whencultured for 20 hours, the cell growth rate and the rate of TNAproduction reached to the maximum levels. The final cell concentrationreached to 75 in absorbance at 600 nm in 24 hours, and the maximum TNAactivity reached to 3.450 units/ml. The TNA production level increased9.2-folds that of the production level obtained when the cells harboringplasmids with lac-promoters were cultured in LB medium under inducingconditions for IPTG.

After SDS-PAGE, the protein band was analyzed using a scanningdensitometer. As a result, the content of the soluble enzyme was deducedas approximately 40% of the total cellular proteins in the extract ofEscherichia coli. Most of the TNA expressed in the cells were producedin the soluble form.

The specific activity for L-tryptophan synthesis in the crude extract ofEscherichia coli HB101/pHCE19T(II)-TNA was approximately 0.15 units/mgcells. This result suggests that the thermostable enzyme includingthermostable tryptophan indole-lyase can be expressed efficiently by theconstitutive promoter.

EXAMPLE 6 Effects of Various Reaction Conditions on Production ofL-Tryptophan

1) Temperature

In order to study the thermal stability of TNA, TNA was incubated atvarious temperatures for 30 minutes in 50 mM potassium phosphate buffer(pH 8.5), and thereafter remaining activities were determined. An enzymesolution obtained after heat treatment of a cell-free extract at 60° C.was used as a biocatalyst of this experiment.

As shown in FIG. 11, this enzyme was stable up to 60° C., and 95% of theenzyme activity thereof was maintained after the heat treatment. Sincethe stability of TNA is thought to be a main factor for determining theproductivity in the synthesis of L-tryptophan, TNA is considered to haveexcellent possibilities in improving the enzymatic method. In addition,the maximum reaction temperature for synthesizing L-tryptophan was 65°C.

2) pH

The effect of pH on the rate of the L-tryptophan synthesis by TNA wasstudied in the presence of excess ammonium chloride and sodium pyruvate.The reaction was carried out in each of buffers having various pH's. Asshown in FIG. 11, the effect of pH on the synthesis activity of TNA isconsiderably significant, and the maximum activity of TNA forL-tryptophan synthesis was found at pH 9.0. In glycine/NaOH buffer,there was observed drastic inhibition to the enzyme reaction.

3) Ammonium Donor

In order to select the most suitable ammonium donor in the synthesis ofL-tryptophan, for a reaction mixture, there were used various ammoniumsalts such as ammonium acetate, ammonium chloride, ammonium citrate,ammonium formate, ammonium nitrate, ammonium oxalate, diammoniumhydrogenphosphate, and ammonium sulfate. The results are shown in Table1.

TABLE 1 Ammonium Donor Relative Activity (%) Ammonium acetate 85.8Ammonium chloride 100 Ammonium formate 58.8 Diammonium hydrogenphosphate82.4 Ammonium nitrate 62.3 Diammonium sulfate 80.4 Reaction Conditions:5 mM or 25 mM indole, 200 mM sodium pyruvate, 1.0M ammonium donor,pH9.0, 60° C.

As shown in Table 1, ammonium chloride was the most suitable ammoniumdonor in the synthesis of L-tryptophan. The tryptophan synthesizingactivity of TNA in the case where ammonium acetate or diammoniumhydrogenphosphate was used was about 80% of the activity when ammoniumchloride was used. In addition, the effect of ammonium chloride in theconcentration range of 0–1.5 M on the synthesis of L-tryptophan wasshown in FIG. 12 panel (A). The synthesizing activity was largelydependent on the concentration of ammonium chloride and had almost thesame level as that of 1.0–1.5 M ammonium chloride.

4) Indole Concentration

Since thermostable enzymes tend to be stable toward chemical-modifyingagents, there can be provided remarkable advantages in biotechnologicalprocesses using enzymes as a biocatalyst. In order to evaluate thestability of the TNA, the effect of the indole concentration on theproductivity of thermostable TNA was investigated. As shown in FIG. 12panel (B), although the tryptophan synthesizing activity of TNA wasincreased to an indole concentration of 5 mM, the activity was decreasedin stages after exceeding the concentration because of the toxicity ofindole. Concurrently, TNA of Escherichia coli was completely inactivatedat 3 mM of indole. This result indicates that those of Symbiobacteriumtoebii has a relatively high stability for inactivation of enzyme byindole, as compared with those of Escherichia coli.

5) Concentration of Sodium Pyruvate

FIG. 12 panel (C) shows the results of studying the L-tryptophansynthesizing activities by TNA at a variety of concentrations within therange of from 0 to 300 mM of sodium pyruvate. The rate of tryptophanproduction was linearly increased in proportion to the concentration ofsodium pyruvate. The maximum synthesizing activity was obtained at apyruvate concentration of 0.1 M. After exceeding the concentration, therate of tryptophan synthesis was kept at a similar value.

6) Triton™ X-100

Nonionic surfactants such as Triton X-100 form micelles to includeindole therein and thus function as reservoirs for indole during theenzyme reaction for the synthesis of L-tryptophan. Therefore, theeffects of Triton X-100 on the rate of L-tryptophan production werestudied and the production rate was compared with the production rate ina reaction mixture without Triton X-100. The results are shown in FIG.13. In a reaction mixture with 3% (v/v) Triton X-100, the maximum rateof L-tryptophan synthesis was obtained at an indole concentration of 50mM which was 10 times higher than that of a reaction mixture withoutTriton X-100. After exceeding the concentration, the productivity ofL-tryptophan was reduced.

7) Optimization of Pre-Treatment of Biocatalysts for Production ofL-Tryptophan

In order to compare the efficiencies of various biocatalysts on theproduction of L-tryptophan, a biocatalyst containing a cell-freeextract, intact whole cells, penetrated cells and acetone-dried cellswere added to a reaction mixture containing 5 mM or 25 mM indole. Thepenetrated cells were prepared by treating the whole cells with tolueneor methanol and then centrifuging. The acetone-dried cells were obtainedby treating the whole cells with acetone and subsequently drying thecells with air-stream. The concentrations of sodium pyruvate andammonium chloride were 200 mM and 1 M, respectively.

TABLE 2 Relative Relative Activity (%) Activity (%) Type of Biocatalysts(5 mM indole) (25 mM indole) Whole intact cells 100 100 Cell-freeextract 98.2 75.5 Cell treated with 5% methanol 98.5 78.9 Cell treatedwith 10% methanol 97.5 71.7 Cell treated with 5% toluene 95.5 69.9 Celltreated with 10% toluene 90.2 53.3 Cell dried with acetone 97.5 62.4Reaction conditions: 5 mM or 25 mM indole, 200 mM sodium pynivate, 1.0Mammonium chloride, pH9.0, 37° C.

As shown in Table 2, L-tryptophan was effectively synthesized in all thebiocatalysts tested at a low concentration of indole (5 mM).Concurrently, only the intact cells showed a high L-tryptophansynthesizing activity at a high concentration of indole (25 mM). In thiscase, other biocatalysts of a lower activity seem to be caused by theinhibition of enzyme through the direct interaction between the enzymeand high concentration of localized indole. The results show that thewhole cells are the most suitable biocatalyst for an efficientproduction of L-tryptophan. Therefore, the whole cells in which TNA wasexcessively produced were used as a biocatalyst for the production ofL-tryptophan.

8) Production of L-Tryptophan by Whole Cell Biocatalyst

Pyruvate as a substrate is very unstable at a temperature higher than45° C., and at this temperature, pyruvate immediately disappears in areaction mixture as deduced by reduction via NADH in the presence oflactate dehydrogenase. The formation of imines with ammonia was probablythe main reaction responsible to a decrease in pyruvate. However, itcould not remove the occurrence of a further reaction of pyruvate.Because of the thermal instability of pyruvate, the synthesis reactioncannot stably proceed at 65° C. Therefore, large-scale quantity ofL-tryptophan could not be produced. Accordingly, the production ofL-tryptophan was performed at 37° C. in 1 liter of the reaction mixturein an enzyme reactor.

The starting reaction mixture comprises 1.0 M ammonium chloride, 100 mMsodium pyruvate, 30 mM indole, 0.1 mM PLP, 0.1% sodium sulfite and 10 g(dried weight)/l of Escherichia coli HB101/pHCE19T(II)-TNA cells. 3%Triton X-100 was added to the reaction mixture, and the added indole wassolubilized. In order to prevent depletion of the substrate, indole andsodium pyruvate were continuously supplied to the reaction solution.

FIG. 14 shows a reaction pattern of the production of L-tryptophan inthe enzyme reactor. The rate of L-tryptophan production was maintainedat the same level for 27 hours, and then gradually decreased. By theanalysis of the reaction mixture by means of HPLC, this reduction of therate of L-tryptophan production was found to be caused by the inhibitiondue to the indole accumulated in a large amount in the reactor. Fromthree hours after starting the reaction, L-tryptophan began toprecipitate as a white crystal, whereby the reaction was allowed toefficiently proceed. Twenty-four hours after starting the reaction,approximately 66.2 g/l (0.325 M) of L-tryptophan was produced at 37° C.from the indole at a conversion efficiency of 85%.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1 shows a sequence of a promoter region in pHCE19T(II).

SEQ ID NO: 2 shows a sequence of a promoter region in pHCE19(II).

SEQ ID NO: 3 shows a sequence of pHCE19T(II).

SEQ ID NO: 4 shows a sequence of pHCE19(II).

SEQ ID NO: 5 shows a sequence of an oligonucleotide sequence foramplifying a promoter of D-AAT gene derived from Bacillus sp.

SEQ ID NO: 6 shows a sequence of an oligonucleotide sequence foramplifying a promoter of D-AAT gene derived from Bacillus sp.

SEQ ID NO: 7 shows a sequence of an oligonucleotide sequence foramplifying a promoter of D-AAT gene derived from Bacillus sp.

SEQ ID NO: 8 shows a sequence of an oligonucleotide sequence foramplifying TNA gene derived from Symbiobacterium toebii SC-1.

SEQ ID NO: 9 shows a sequence of an oligonucleotide sequence foramplifying TNA gene derived from Symbiobacterium toebii SC-1.

INDUSTRIAL APPLICABILITY

According to the DNA of the present invention, since the DNA has apromoter sequence capable of constitutively expressing a desired geneproduct, without inducing by an inducer, there are exhibited excellenteffects that the desired gene product can be expressed easily andinexpensively at a high level. Therefore, the DNA of the presentinvention allows to produce an expression product for a useful gene inindustrial scale. In addition, the method for producing a protein of thepresent invention allows to produce an expression product for a usefulgene in industrial scale.

1. An isolated DNA, wherein the isolated DNA exhibits a constitutivepromoter activity in Escherichia coli or a bacterium belonging to thegenus Bacillus, and wherein the isolated DNA has a nucleotide sequenceselected from the group consisting of: (A) the nucleotide sequence ofSEQ ID NO: 1; and (B) a nucleotide sequence having at least 80% sequenceidentity to the nucleotide sequence of SEQ ID NO:
 1. 2. The isolated DNAaccording to claim 1, wherein the isolated DNA is capable of expressinga foreign gene in the absence of an inducer, when located upstream fromthe gene.
 3. A recombinant DNA comprising the DNA of claim 1 and aforeign gene, wherein the foreign gene is operably located.
 4. Therecombinant DNA according to claim 3, wherein the foreign gene is anucleic acid selected from the group consisting of a nucleic acidencoding a protein, a nucleic acid encoding antisense RNA, and a nucleicacid encoding a ribozyme.
 5. A gene expression vector, at leastcomprising the DNA of claim
 1. 6. The gene expression vector accordingto claim 5, wherein the vector is a vector selected from the groupconsisting of plasmid vectors, phage vectors and viral vectors.
 7. Thegene expression vector according to claim 5 or 6, which comprises thenucleotide sequence of SEQ ID NO:
 3. 8. An expression vector comprisingthe recombinant DNA of claim
 3. 9. The expression vector according toclaim 8, wherein the vector is a vector selected from the groupconsisting of plasmid vectors, phage vectors and viral vectors.
 10. Atransformant of Escherichia coli or a Bacillus bacterium having therecombinant DNA of claim 3 or
 4. 11. A method for producing a protein,comprising: culturing the transformant of claim 10, and collecting aprotein from the resulting culture.
 12. A kit for producing a protein,at least comprising DNA of claim 1 or 2, or a gene expression vector atleast comprising the DNA of claim 1 or
 2. 13. A transformant ofEscherichia coli or a Bacillus bacterium having the expression vector ofclaim 8 or 9.